Enhanced Effects of Intermittent Fasting by Magnetic Fields in Severe Diabetes

Intermittent fasting (IF) is a convenient dietary intervention for multiple diseases, including type 2 diabetes. However, whether it can be used as a long-term antidiabetic approach is still unknown. Here, we confirm that IF alone is beneficial for both moderate and severe diabetic mice, but its antidiabetic effects clearly diminish at later stages, especially for severe diabetic db/db mice, which have obviously impaired autophagy. We found that static magnetic fields can directly promote actin assembly and boost IF-induced autophagy. Consequently, the pancreatic islet and liver were improved, and the antidiabetic effects of IF were boosted. In fact, at later stages, combined static magnetic field and IF could reduce the blood glucose level of moderate type 2 diabetic mice by 40.5% (P < 0.001) and severe type 2 diabetes by 34.4% (P < 0.05), when IF alone no longer has significant blood glucose reduction effects. Therefore, although IF is generally beneficial for diabetes, our data reveal its insufficiency for late-stage diabetes, which can be compensated by a simple, noninvasive, long-lasting, and nonpharmacological strategy for effective long-term diabetic control.

Similar immunofluorescence staining was performed for apoptosis marker TUNEL and insulin.The TUNEL assay was used to detect apoptotic cells in pancreatic tissues using the TUNEL detection kit (C1098, Beyotime, China), which labels dUTP with a nickel end.
3,3'-diaminobenzidine (DAB) reagent was used for staining, and the samples were incubated at room temperature for 5-30 minutes.Image J software was used to count TUNEL-positive cells in the insulin-labeled regions and quantitatively analyze the number of apoptotic β-cells in each islet.

DHE staining
DHE was dissolved in DMSO to detect ROS in mice liver and islets.Paraffin sections of liver and pancreatic islets after dewaxing were taken and treated with PBS and tissue autofluorescence quencher for 10 minutes.Then, 100 μl of staining working solution (DHE diluted at a ratio of 1:1000) was added, and the sections were incubated in the dark at 37 ℃ for 60 minutes.Finally, the sections were stained with 4',6-diamidino-2-phenylindole (DAPI) and sealed with an anti-fluorescence quencher.
Subsequently, the cells were stained with 300 nM 4',6-diamidino-2-phenylindole (DAPI) at room temperature for 5 minutes and then fixed with Antifade Pro-Long Gold (P36980, Invitrogen).Both Min6 and RPE1 cells were seeded on 35 mm culture dishes containing coverslips and allowed to adhere for 24 h.After treating the cells with 100 nM cytochalasin D (cytoD) for 2 h, the culture medium was removed, and the cells were washed with PBS before changing to normal culture medium.Samples were collected at 3 h and 6 h after treatment.
The cells on coverslips were washed with PBS, fixed with 4% paraformaldehyde at room temperature for 20 minutes, and then blocked with Abdil-TX (0.1% Triton X-100, 2% bovine serum albumin, 0.05% NaN 3 ) for 30 minutes.F-actin was stained with Alexa Fluor™594 phalloidin (A12381, Invitrogen) at room temperature for 0.5 h.Next, the cells were stained with 300 nM 4',6-diamidino-2-phenylindole (DAPI) for 5 minutes at room temperature, followed by fixing with Antifade Pro-Long Gold (P36980, Invitrogen).The images were captured using an Olympus fluorescence microscope (SpinSR10, Olympus, Tokyo, Japan).The length of F-actin filaments in cells were measured using Image J software.

Western blotting
Min6 cells (3×10 5 cells/mL) were seeded into 35 mm culture dishes and allowed to adhere to the walls.Subsequently, the cells were subjected to either regular culture or intermittent serum starvation.Simultaneously, they were exposed to sham, SMF#1 or SMF#2 for 36 h.

Electron microscope analysis
After intermittent starvation and SMF treatment, Min6 cells were collected and fixed in 3% glutaraldehyde (containing 2% paraformaldehyde) at room temperature for 1 h, followed by overnight incubation at 4 °C and subsequent washing with PBS.The cells were then fixed twice in PBS containing 1% osmium tetroxide and 1.5% potassium ferrocyanide at room temperature and exposed to 2% uranyl acetate solution.After PBS rinsing, the cells underwent graded ethanol dehydration and were embedded in Spurr low viscosity media.
The samples were polymerized overnight at 70 °C.Using Leica Ultra CUT UC7, 100 nm ultrathin sections were cut onto copper grids.After staining with 0.2% lead citrate, the ultrathin sections were observed using a 120 KV electron microscope (Tecnai G2 SPIRIT BioTWIN, FEI Corporation, RRID:SCR_021365).
The samples were then subjected to either Sham or SMF treatment for 5 min at room temperature under GMF conditions before TEM measurement.For transmission electron microscopy (TEM) experiments, the samples were added to a 200-mesh grid (20 s for ion sputtering) and incubated for 90 s.After grid drying, 1% uranyl acetate was added and incubated for 90 s.The grid was then stained three times with dye, air-dried, and imaged by a electron microscope operated at 200 kV (Talos F200X, FEI Corporation, RRID:SCR_019907).

Open field test (OFT)
The open-field test apparatus (SA215, SANS, China, RRID:SCR_015938) consists of a white plastic board (1000 × 1000 × 400 mm), divided into four enclosed square spaces, allowing simultaneous testing of 4 mice at a time.The enclosed open field is partitioned into a central zone and a peripheral zone using 25 beams of infrared arrays, with the central area defined by the central 9 squares.At the beginning of the experiment, the mice are gently placed in the center of the square space and allowed to freely explore the apparatus for 5 minutes.The entire activity is recorded by a camera installed at the top of the device and connected to a computer, with synchronized data collected and analyzed using the ANY-Maze video tracking system (Stoelting, USA, RRID:SCR_014289) installed on the computer.After each trial, the open-field apparatus is wiped clean with 75% ethanol.

Mechanical withdrawal threshold (MWT) test
The mechanical withdrawal threshold of db/db mice was measured using a Von Frey aesthesiometer (IITC, USA, RRID:SCR_021751).Each mouse was placed individually in a testing chamber and allowed to adapt to the environment freely for 20 minutes.The Von Frey aesthesiometer was used to stimulate the left hind paw of the mouse.Each mouse underwent 5 repetitions of the stimulus, and the force required for the mouse to rapidly withdraw its paw in response to the stimulus was recorded.At least 5 minutes of rest was given between each stimulation.The cell culture dishes were placed above the north (N) or south (S) pole of the magnets, which provide SMF#1 and SMF#2.The control group was placed inside the same cell culture incubator but far away from the magnets.The measured magnetic flux density of control group is ~0.0001T, which is ~5000 times lower than the SMF group using a 0.5 T SMF.

Fig. S4 .
Fig. S4.Static magnetic field combined with intermittent fasting reduces hepatic lipid accumulation and multiple organ lesions in db/db mice.(A) Organ coefficient of db/db mice (It represents the ratio of the organ weight to the weight of the whole body, n = 3-9 mice/group).(B) Representative HE images of mice liver, kidney, retina, hippocampus and muscle (The box area is partially enlarged as shown in Figure 4D, n = 3 mice/group).(C) Mechanical withdrawal threshold (MWT) of db/db mice (n = 6-9 mice/group).All data are

Fig. S5 .
Fig. S5.SMF+IF have a tendency to improve the exercise and exploration ability in db/db diabetic mice.(A) The open field test (OFT) related indicators (n = 6-9 mice/group).(B) The OFT heatmap (n = 6-9 mice/group).All data are presented as mean ± SEM and analyzed by GraphPad Prism 9.0.

Fig. S10 .
Fig. S10.LC3B immunofluorescence staining of Min6 cells and actin immunofluorescence staining of RPE1 cells.(A) LC3B immunofluorescence staining of min6 cells.Intermittent starvation of Min6 cells using 1% serum starvation was carried out for 48 h with simultaneous SMF treatment for 36 h.(Scale bar: 20 μm).(B) RPE1 cells were treated with 100 nM cytoD for 2 h, with or without additional washout to allow recovery for 3 h with Sham or SMF, before they were fixed and stained with phalloidin

Fig. S11 .
Fig. S11.Cell experimental setup.The cell culture dishes were placed above the north (N) or south (S) pole of the magnets, which provide SMF#1 and SMF#2.The control group was placed inside the same cell culture incubator but far away from the magnets.The measured magnetic flux density of control group is ~0.0001T, which is ~5000 times lower than the SMF group using a 0.5 T SMF.